Many different experiments have shown that the transcription factor Pax6 has a central role in development of the eye. The phenotype of mutant mice and humans indicates that Pax6 is required for development of the eye. The primary goal of the experiments described in this application is the identification of genes that regulate Pax6 and an understanding of how they function in the lens and lacrimal gland (LG) lineages. We will examine this question at a number of different levels. We previously identified a transcriptional enhancer in the Pax6 gene that appears to be required for expression of Pax6 in the embryonic ectoderm that gives rise to the lens and LG epithelium. In the first aim of this application (Aim 14) we will perform a fate-mapping analysis so that we can determine the fate of cells that express Pax6 from the ectoderm enhancer. We will also investigate the function of the ectoderm enhancer by determining whether the boundaries of evolutionarily conserved regions define functional sub-domains (Aim 15). To identify the genes that regulate Pax6 transcription in the lens and LG lineages (Aim 16), we will first use biochemical purification to isolate proteins that bind DNA representing the ectoderm enhancer. Protein identification followed by cDNA and gene cloning will provide the tools for a complete analysis of gene function. We will use mouse gene-targeting methods to determine whether newly identified genes regulate Pax6 in vivo (Aim 17). The gene-targeted mice generated for Aim 16 can also be used for epistasis analysis (Aim 18). This would permit a new Pax6-regulating gene to be placed in existing pathways that describe lens and LG development. The ability of Pax6 to direct the formation of all eye components in a vertebrate embryo may have significant consequences for human health. Cell-based therapies, where a pluripotent or differentiated cell type is produced in vitro and then used to repair damaged or genetically deficient tissue, are being developed and offer considerable promise. In the case of the eye, it is conceivable that the activity of Pax6 might be used to generate mature cell types useful in therapy. Only a complete understanding of the activity and regulation of Pax6 will permit these opportunities to be exploited. The analysis described in this application will take us closer to this goal.